Aberrant lipid accumulation and retinal pigmental epithelium dysfunction in PRCD-deficient mice.

Progressive Rod-Cone Degeneration (PRCD) is an integral membrane protein found in photoreceptor outer segment (OS) disc membranes and its function remains unknown. Mutations in Prcd are implicated in Retinitis pigmentosa (RP) in humans and multiple dog breeds. PRCD-deficient models exhibit decreased levels of cholesterol in the plasma. However, potential changes in the retinal cholesterol remain unexplored. In addition, impaired phagocytosis observed in these animal models points to potential deficits in the retinal pigment epithelium (RPE). Here, using a Prcd -/- murine model we investigated the alterations in the retinal cholesterol levels and impairments in the structural and functional integrity of the RPE. Lipidomic and immunohistochemical analyses show a 5-fold increase in the levels of cholesteryl esters (C.Es) and accumulation of neutral lipids in the PRCD-deficient retina, respectively, indicating alterations in total retinal cholesterol. Longitudinal fundus and spectral domain optical coherence tomography (SD-OCT) examinations showed focal lesions and RPE hyperreflectivity. Strikingly, the RPE of Prcd -/- mice exhibited age-related pathological features such as neutral lipid deposits, lipofuscin accumulation, Bruch's membrane (BrM) thickening and drusenoid focal deposits, mirroring an Age-related Macular Degeneration (AMD)-like phenotype. We propose that the extensive lipofuscin accumulation likely impairs lysosomal function, leading to the defective phagocytosis observed in Prcd -/- mice. Our findings support the dysregulation of retinal cholesterol homeostasis in the absence of PRCD. Further, we demonstrate that progressive photoreceptor degeneration in Prcd -/- mice is accompanied by progressive structural and functional deficits in the RPE, which likely exacerbates vision loss over time.

Astrocyte ensheathment of calyx-forming axons of the auditory brainstem precedes accelerated expression of myelin genes and myelination by oligodendrocytes.

Early postnatal brain development involves complex interactions among maturing neurons and glial cells that drive tissue organization. We previously analyzed gene expression in tissue from the mouse medial nucleus of the trapezoid body (MNTB) during the first postnatal week to study changes that surround rapid growth of the large calyx of Held (CH) nerve terminal. Here, we present genes that show significant changes in gene expression level during the second postnatal week, a developmental timeframe that brackets the onset of airborne sound stimulation and the early stages of myelination. Gene Ontology analysis revealed that many of these genes are related to the myelination process. Further investigation of these genes using a previously published cell type-specific bulk RNA-Seq data set in cortex and our own single-cell RNA-Seq data set in the MNTB revealed enrichment of these genes in the oligodendrocyte lineage (OL) cells. Combining the postnatal day (P)6-P14 microarray gene expression data with the previously published P0-P6 data provided fine temporal resolution to investigate the initiation and subsequent waves of gene expression related to OL cell maturation and the process of myelination. Many genes showed increasing expression levels between P2 and P6 in patterns that reflect OL cell maturation. Correspondingly, the first myelin proteins were detected by P4. Using a complementary, developmental series of electron microscopy 3D image volumes, we analyzed the temporal progression of axon wrapping and myelination in the MNTB. By employing a combination of established ultrastructural criteria to classify reconstructed early postnatal glial cells in the 3D volumes, we demonstrated for the first time that astrocytes within the mouse MNTB extensively wrap the axons of the growing CH terminal prior to OL cell wrapping and compaction of myelin. Our data revealed significant expression of several myelin genes and enrichment of multiple genes associated with lipid metabolism in astrocytes, which may subserve axon wrapping in addition to myelin formation. The transition from axon wrapping by astrocytes to OL cells occurs rapidly between P4 and P9 and identifies a potential new role of astrocytes in priming calyceal axons for subsequent myelination.

Transcriptional profiling reveals roles of intercellular Fgf9 signaling in astrocyte maturation and synaptic refinement during brainstem development.

Neural tissue maturation is a coordinated process under tight transcriptional control. We previously analyzed the kinetics of gene expression in the medial nucleus of the trapezoid body (MNTB) in the brainstem during the critical postnatal phase of its development. While this work revealed timed execution of transcriptional programs, it was blind to the specific cells where gene expression changes occurred. Here, we utilized single-cell RNA-Seq to determine transcriptional profiles of each major MNTB cell type. We discerned directional signaling patterns between neuronal, glial, and vascular-associated cells for VEGF, TGFß, and Delta-Notch pathways during a robust period of vascular remodeling in the MNTB. Furthermore, we describe functional outcomes of the disruption of neuron-astrocyte fibroblast growth factor 9 (Fgf9) signaling. We used a conditional KO (cKO) approach to genetically delete Fgf9 from principal neurons in the MNTB, which led to an early onset of glial fibrillary acidic protein (Gfap) expression in astrocytes. In turn, Fgf9 cKO mice show increased levels of astrocyte-enriched brevican (Bcan), a component of the perineuronal net matrix that ensheaths principal neurons in the MNTB and the large calyx of Held terminal, while levels of the neuron-enriched hyaluronan and proteoglycan link protein 1 (Hapln1) were unchanged. Finally, volumetric analysis of vesicular glutamate transporters 1 and 2 (Vglut1/2), which serves as a proxy for terminal size, revealed an increase in calyx of Held volume in the Fgf9 cKO. Overall, we demonstrate a coordinated neuron-astrocyte Fgf9 signaling network that functions to regulate astrocyte maturation, perineuronal net structure, and synaptic refinement.

Glial Cell Expansion Coincides with Neural Circuit Formation in the Developing Auditory Brainstem.

Neural circuit formation involves maturation of neuronal, glial and vascular cells, as well as cell proliferation and cell death. A fundamental understanding of cellular mechanisms is enhanced by quantification of cell types during key events in synapse formation and pruning and possessing qualified genetic tools for cell type-specific manipulation. Acquiring this information in turn requires validated cell markers and genetic tools. We quantified changing proportions of neurons, astrocytes, oligodendrocytes, and microglia in the medial nucleus of the trapezoid body (MNTB) during neural circuit development. Cell type-specific markers, light microscopy and 3D virtual reality software, the latter developed in our laboratory, were used to count cells within distinct cell populations at postnatal days (P)3 and P6, bracketing the period of nerve terminal growth and pruning in this system. These data revealed a change from roughly equal numbers of neurons and glia at P3 to a 1.5:1 ratio of glia to neurons at P6. PCNA and PH3 labeling revealed that proliferation of oligodendrocytes contributed to the increase in glial cell number during this timeframe. We next evaluated Cre driver lines for selectivity in labeling cell populations. En1-Cre was specific for MNTB neurons. PDGFRα-Cre and Aldh1L1-Cre, thought to be mostly specific for oligodendrocyte lineage cells and astrocytes, respectively, both labeled significant numbers of neurons, oligodendrocytes, and astrocytes and are non-specific genetic tools in this neural system.

Temporal patterns of gene expression during calyx of held development.

Relating changes in gene expression to discrete developmental events remains an elusive challenge in neuroscience, in part because most neural territories are comprised of multiple cell types that mature over extended periods of time. The medial nucleus of the trapezoid body (MNTB) is an attractive vertebrate model system that contains a nearly homogeneous population of neurons, which are innervated by large glutamatergic nerve terminals called calyces of Held (CH). Key steps in maturation of CHs and MNTB neurons, including CH growth and competition, occur very quickly for most cells between postnatal days (P)2 and P6. Therefore, we characterized genome-wide changes in this system, with dense temporal sampling during the first postnatal week. We identified 541 genes whose expression changed significantly between P0-6 and clustered them into eight groups based on temporal expression profiles. Candidate genes from each of the eight profile groups were validated in separate samples by qPCR. Our tissue sample permitted comparison of known glial and neuronal transcripts and revealed that monotonically increasing or decreasing expression profiles tended to be associated with glia and neurons, respectively. Gene ontology revealed enrichment of genes involved in axon pathfinding, cell differentiation, cell adhesion and extracellular matrix. The latter category included elements of perineuronal nets, a prominent feature of MNTB neurons that is morphologically distinct by P6, when CH growth and competition are resolved onto nearly all MNTB neurons. These results provide a genetic framework for investigation of general mechanisms responsible for nerve terminal growth and maturation.

Maturation of synaptic partners: functional phenotype and synaptic organization tuned in synchrony.

Maturation of principal neurons of the medial nucleus of the trapezoid body (MNTB) was assessed in the context of the developmental organization and activity of their presynaptic afferents, which grow rapidly to form calyces of Held and to establish mono-innervation between postnatal days (P)2 and 4. MNTB neurons and their inputs were studied from embryonic day (E)17, when the nucleus was first discernable, until P14 after the onset of hearing. Using a novel slice preparation containing portions of the cochlea, cochlear nucleus and MNTB, we determined that synaptic inputs form onto MNTB neurons at E17 and stimulation of the cochlear nucleus can evoke action potentials (APs) and Ca(2+) signals. We analysed converging inputs onto individual MNTB neurons and found that competition among inputs was resolved quickly, as a single large input, typically larger than 4 nA, emerged from P3-P4. During calyx growth but before hearing onset, MNTB cells acquired their mature, phasic firing property and quantitative real-time PCR confirmed a coincident increase in low threshold K(+) channel mRNA. These events occurred in concert with an increase in somatic surface area and a 7-fold increase in the current threshold (30 to >200 pA) required to evoke action potentials, as input resistance (R(in)) settled from embryonic values greater than 1 GΩ to approximately 200 MΩ. We postulate that the postsynaptic transition from hyperexcitability to decreased excitability during calyx growth could provide a mechanism to establish the mature 1:1 innervation by selecting the winning calyceal input based on synaptic strength. By comparing biophysical maturation of the postsynaptic cell to alterations in presynaptic organization, we propose that maturation of synaptic partners is coordinated by synaptic activity in a process that is likely to generalize to other neural systems.

The micro-architecture of mitochondria at active zones: electron tomography reveals novel anchoring scaffolds and cristae structured for high-rate metabolism.

Mitochondria are integral elements of many nerve terminals. They must be appropriately positioned to regulate microdomains of Ca(2+) concentration and metabolic demand, but structures that anchor them in place have not been described. By applying the high resolution of electron tomography (ET) to the study of a central terminal, the calyx of Held, we revealed an elaborate cytoskeletal superstructure that connected a subset of mitochondria to the presynaptic membrane near active zones. This cytoskeletal network extended laterally and was well integrated into the nerve terminal cytoskeleton, which included filamentous linkages among synaptic vesicles. ET revealed novel features of inner membrane for these mitochondria. Crista structure was polarized in that crista junctions, circular openings of the inner membrane under the outer membrane, were aligned with the cytoskeletal superstructure and occurred at higher density in mitochondrial membrane facing the presynaptic membrane. These characteristics represent the first instance where a subcomponent of an organelle is shown to have a specific orientation relative to the polarized structure of a cell. The ratio of cristae to outer membrane surface area is large in these mitochondria relative to other tissues, indicating a high metabolic capacity. These observations suggest general principles for cytoskeletal anchoring of mitochondria in all tissues, reveal potential routes for nonsynaptic communication between presynaptic and postsynaptic partners using this novel cytoskeletal framework, and indicate that crista structure can be specialized for particular functions within cellular microdomains.